manual of methods for determining micronutrients in fortified foods

V. Reagents
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Acetonitrile (CH 3
CN), mol wt. 41.05, HPLC grade.
Calcium hydroxide. (Ca(OH) 2
), mol wt. 74.09, p.a
Ethanol.
o-Phosphoric acid (H 3
PO 4
), 85% purity, mol wt. 98.00, p.a.
Oxalic acid (C 2
H 2
O 4
.2H 2
O), mol wt. 126.07, p.a.
Sodium dodecylsulfate (CH 3
(CH 2
) 11
OSO 3
Na), ≥ 99% purity for ion-pair chromatography, mol wt.
288.38. Fluka 71726.
Mobile phase:
Solution A (acidified water): Dilute 1.00 mL o-phosphoric acid in 600 mL water and filter through
0.45 um.
Solution B: In a 500 mL beaker, mix 120 mL water and 230 mL acetonitrile. Dissolve 1.00 g sodium
dodecyl sulfate in this solution. Bring total volume to 400 mL with water. Filter through 0.45 um.
Mix solution A and B to make 1L mobile phase. Mix well.
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Stock niacin standard solution (2000 mg/L):
Weigh 200 mg niacin and dissolve in ethanol:water (1+1) in a 100 mL volumetric flask. Bring to
volume with the mixture ethanol:water (1+1) and mix well. This solution is stable at least 2 weeks
at room temperature.
Working Standard solution-2 mg/L:
Take 5 mL of stock standard solution and dilute to 100 mL with water. Then take 2 mL of the
dilution and dilute to 100 mL with water.
VI. Procedure
1. Weigh 2 g flour in a 500 mL blender jar. The sample weight should contain about 0.04 mg niacin.
2. Add 198 mL water and add 10 g calcium hydroxide.
3. Standard treatment: Prepare three standard solutions with different concentrations.
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Low standard: Pipet 10 mL of the working standard solution (2 mg/L) into 500 mL
Erlenmeyer flask containing 190 mL water.
Intermediate standard: Pipet 20 mL of the working standard solution into 500 mL Erlenmeyer
flask containing 180 mL water.
High standard: Pipet 40 mL of the working standard solution into 500 mL Erlenmeyer flask
containing 160 mL water.
Add 10 g calcium hydroxide and treat the standard as the sample starting from step 4.
4. Blend aprox. 30 seconds at high speed using a vertical stirrer or blender.
5. Autoclave 15 min at 121°C.
6. Cool in ice bath at least 30 min.
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